A. Keith Stobart

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the ω3/6 Δ8-desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Δ9-specific elongating activity from Isochrysis galbana, a Δ8-desaturase from Euglena gracilis and a Δ5-desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Δ9-elongating activity, which may bypass rate-limiting steps present in the conventional Δ6-desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

Identification of a cDNA encoding a novel C18-Δ9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana 1

Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n‐3, Δ4,7,10,13,16,19). We used a polymerase chain reaction‐based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid‐elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18‐Δ9 polyunsaturated fatty acids, linoleic acid (C18:2n‐6, Δ9,12) and α‐linolenic acid (C18:3n‐3, Δ9,12,15), to eicosadienoic acid (C20:2n‐6, Δ11,14) and eicosatrienoic acid (C20:3n‐3, Δ11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n‐3, Δ5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ8 desaturation pathway.

Functional identification of a fatty acid Δ5 desaturase gene from Caenorhabditis elegans

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid Δ5 desaturase. Saccharomyces cerevisiae expressing the full‐length cDNA was able to convert di‐homo‐γ‐linolenic acid to arachidonic acid, thus confirming Δ5 desaturation. The 1341 bp Δ5 desaturase sequence contained an N‐terminal cytochrome b 5 domain and was located within a kilobase of the C. elegans Δ6 desaturase on chromosome IV. With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication. This is the first example of a Δ5 desaturase gene isolated from an animal.

Plant desaturases: harvesting the fat of the land.

The past few years have witnessed a major upsurge in research towards the goal of modifying the lipid composition of plants. Genes encoding a range of different fatty acid desaturase activities have been cloned, and the evolutionary relationships between and within different classes of enzymes have tentatively been established. The effects of expressing some of these desaturases in heterologous hosts have also been studied, often producing unexpected results which contribute further to our understanding of plant lipid modification. It is to be hoped that, in the near future, the goal of producing unusual and valuable fatty acids in transgenic oilseeds will be achieved on a commercial scale.

Distribution and biosynthesis of stearidonic acid in leaves of Borago officinalis

Abstract An octadecatetraenoic acid was present as a major fatty acid component of the leaf lipids of borage. Gas chromatography-mass spectrometry of its picolinyl esters gave unsaturation centres at carbons Δ 6 , Δ 9 , Δ 12 , and Δ 15 and confirmed its identity as stearidonic acid (SDA; octadecatetraenoic acid, C18:4, Δ 6,9,12,15 . The chloroplast galactolipids, monogalactosyldiacyglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were particularly rich in SDA. SDA was absent, howver, from the plastid phospholipid, phosphatidylglycerol (PG). Stereochemical analysis of the fatty acids in leaf MGDG and phosphatidylcholine (PC) showed that both SDA and γ-linolenic acid (GLA) were almost exclusively located at carbon sn -2 of these complex lipids. In time-course studies with excised seed cotyledons induced to green by light treatment, SDA appeared in the galactolipids before its detection in PC suggesting that its major site of synthesis in the leaf was prokaryotic and largely located in the chloroplasts. Borage seed microsomes, which have high Δ 6 desaturase ( Δ 6 des) activity, catalysed the synthesis of SDA from exogenously supplied α-linolenic acid (ALA). Linseed cotyledons which have an active Δ 15 des, on the other hand, could not convert exogenously supplied GLA to SDA. These observations suggest that SDA is formed from ALA via Δ 6 des activity at carbon sn-2 of MGDG and not from GLA and subsequent Δ 15 -desaturation.

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied 14C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [14C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [14C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [14C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [14C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.

The variant ‘his‐box’ of the C18‐Δ9‐PUFA‐specific elongase IgASE1 from Isochrysis galbana is essential for optimum enzyme activity

IgASE1, a C18‐Δ9‐polyunsaturated fatty acid‐specific fatty acid elongase component from Isochrysis galbana, contains a variant histidine box (his‐box) with glutamine replacing the first histidine of the conserved histidine‐rich motif present in all other known equivalent proteins. The importance of glutamine and other variant amino acid residues in the his‐box of IgASE1 was determined by site‐directed mutagenesis. Results showed that all the variation in amino acid sequence between this motif in IgASE1 and the consensus sequences of other elongase components was required for optimum enzyme activity. The substrate specificity was shown to be unaffected by these changes suggesting that components of the his‐box are not directly responsible for substrate specificity.

Synthesis of storage reserves in developing seeds of sunflower

Abstract A combination of biochemical analyses and Northern blotting were used to determine the patterns of storage reserve (oil, 2S albumin, helianthinin, oleosin) synthesis during the early stages of embryo development in sunflower. This demonstrated that oleosin biosynthesis is initiated early in development which is consistent with a role in oil body biogenesis. The patterns of synthesis and accumulation of storage compounds were compared in young developing embryos of sunflower. Oil biosynthesis was clearly initiated very early in development with total lipids accounting for about 18% of the dry weight at the earliest stage studied. This was associated with the accumulation of oleosins and the expression of cytochrome b 5 . In contrast, significant amounts of helianthinin (11S globulin) and 2S albumin were only observed in stages 3 and 4, respectively. The early expression of oleosins contrasts with previous studies of other oilseeds and is consistent with the hypothesis that they are intimately associated with the biogenesis of oil bodies.

Effect of n-6 Polyunsaturated Fatty Acids on Growth and Lipid Composition of Neoplastic and Non-Neoplastic Canine Prostate Epithelial Cell Cultures

Polyunsaturated fatty acids (n‐6) are reported to selectively kill malignant cells. Most investigations, however, did not compare neoplastic with non‐neoplastic cells from the same tissue type. Here we evaluate the effects of n‐6 fatty acids on a non‐neoplastic epithelium cell line (CAPE) and a spontaneous carcinoma cell line (CPA) derived from the canine prostate.

Characterization of a sunflower seed albumin which associates with oil bodies

Abstract An Mr 22 500 protein was purified from isolated oil bodies from sunflower cotyledons. N-terminal amino acid sequencing showed that this protein belonged to the 2S albumin group of storage proteins. A corresponding cDNA clone encoded a preproprotein, comprising two mature 2S albumin proteins one of which corresponded to the oil-body associated albumin. In contrast, the second albumin encoded by the cDNA was related to components purified from a total albumin fraction by RP-HPLC.